Shiga toxin (Stx)-producing E. coli (STEC) is one of the most important pathogens that infected broiler chickens and cause large economic loses in Egypt broiler farms. The present study was designed to determine the prevalence and identification of Non – O157 Shiga toxin (Stx)-producing E. coli in broiler chickens as a hazard in broiler farms. For that a total of 200 cases of broiler chicken samples (lung, liver, heart and gall bladder) were collected from100 freshly dead birds and 100 diseased birds suffering from chronic respiratory disease (CRD) or colibacillosis, from 10 broiler farms showing high mortality ratesin Bohera Governorate, and analyzed in bacteriology laboratory by cultured in some different bacteriological media followed by gram's staining, biochemical tests, serological tests and Polymerase Chain reaction (PCR). The PCR was performed by targeting Shiga toxin producing genes (Stx), (Stx1 and Stx2) in E. coli. E. coli was isolated from 40 (40%) cases in diseased birds, 66 (66%) cases in freshly dead birds, and 106 (53%) cases in both diseased and freshly dead birds. The E. coli prevalence was higher in freshly dead broiler chickens than diseased broiler chickens. In both diseased and freshly dead broiler chickens, the E. coli prevalence was higher in lung samples 41/200 (20.5%), followed by liver samples 30/200 (15%), then heart samples 20/200 (10%), and the gall bladder samples were the lowest prevalence15/200 (7.5%). Serodiagnosis of 10 random selected E. coli strains resulted in ; 2 serotypes O119: H6, 1 serotypeO1:H7, 3 serotypes O146:H21 and 4 serotypes O78. These 10 serotyped strains were further detected by PCR for the (Stx) genes and revealed that, only one strain O146:H21 was positive for the(Stx1)gene, and two strains O78 were positive for the (Stx2)gene. Our current study concluded that, broiler chickens may act as a source of Shiga toxin-producing E. coli (STEC) infection (Stx1 and Stx2), which may leading to mortalities in broiler chickens and cause large economic loses.